PROJECT SUMMARY Port Wine Stain (PWS) is a congenital, progressive vascular malformation of human skin which occurs in an estimated incidence of 3-5 infants per 1,000 live births. Approximately 1.2 million individuals in the United States have PWS birthmarks. Pulsed dye laser (PDL) remains the choice of treatment for PWS. However, only less than 10% of patients achieve complete lesion fading after PDL. Inadequate PWS therapeutic outcome is a clinically significant problem that requires an urgent solution. The etiology and pathogenesis of PWS are currently poorly understood. This knowledge gap is a major obstacle for developing any new effective treatments. PWS is one of the research areas under the NIAMS long-range plan listed as ?understanding the cause of skin vasculature birth defects (e.g., hemangioma and port wine stain) and developing effective therapies.? The studies proposed herein are specifically directed towards understanding pathological mechanisms of PWS and facilitating the development of new, more effective approaches to treatment. In searching for causative substances for PWS, we have identified that circulating CD31+ exosomes, which are derived from human lesional dermal microvascular endothelial cells (hDMVECs), facilitate the formation of vascular phenotypes of PWS. Analysis of the protein content of the serum CD31+ exosomes reveals that PWS CD31+ exosomes harbor a unique molecular milieu that include EphB1, ephrinB2 (EfnB2), a-disintegrin-and- metalloprotease domain 30 (ADAM30), and exocytosis mediator synatotagamin like-1 (SYTL1). Functional analysis shows ADAM30 functions as a sheddase to cleave EfnB2 and its activity is regulated by vascular endothelial growth factor receptor 2 (VEGFR2) signaling. More interestingly, compared to normal controls, PWS serum CD31+ exosomes and hDMVEC culture-derived EphB1/EfnB2/ADAM30 exosomes possess much greater angiogenesis activity. They greatly increased the number and size of new blood vessels formed in a nude mouse xenograft model in vivo. These novel findings motivate us to test a hypothesis that hDMVEC- derived CD31+ exosomes enrich at lesional sites and cause the development and progression of PWS via disruption of cell-cell and cell-ECM interactions and EC barrier function through a molecular pathway involving exosomal EphB1/EfnB2/ADAM30 (Fig. 1). These exosomes are also released into the circulatory system where they can be identified and serve as potential serum biomarkers. In Aim 1, we will determine the specific molecular profiles of CD31+ exosomes derived from PWS ECs as compared to normal controls in vitro and in vivo. We will determine if secretion of CD31+ exosomes from PWS hDMVECs is regulated by exocytosis genes SYTL1 and synatotagamin-1 (SYT1) in vivo and in vitro; In Aim 2, we will determine the mechanisms by which EphB1/EfnB2 regulates VEGFR2 signaling to activate ADAM30 leading to subsequent EfnB2 cleavage; in Aim 3, we will determine if PWS CD31+ exosomal EphB1/EfnB2/ADAM30 induces the formation of PWS vascular phenotypes in vitro; and in Aim 4, we will determine if PWS CD31+ exosomal EphB1/EfnB2/ADAM30 induces angiogenesis and formation of PWS vascular phenotypes in vivo. The long- term goal of this research is to investigate the pathogenesis of PWS for developing new treatments of PWS. The immediate goal is to establish the role of lesional endothelial exosomes in the vascular phenotypes in the pathogenesis of PWS. Our proposed studies are totally novel and data so obtained will be important since they will define a novel molecular pathway for the pathogenesis of PWS and open new avenues for the development of more effective therapeutic strategies for PWS.